Articular cartilage has a low propensity for self-repair, due to which 27 million people are affected by osteoarthritis every year in North America. The current repair techniques used for cartilage defects possess flaws that reduce long-term clinical success. Tissue engineering carries with it the promise of engineering hyaline-like cartilage with physical and biochemical properties, similar to that of native cartilage.

This being said, the primary objective of my project was to engineer clinically relevant sized articular cartilage constructs. To achieve my objective, first, I investigated the effect of continuous culture on cartilaginous tissue growth. Constructs grown under continuous media flow significantly accumulated more collagen and glycosaminoglycan, and displayed a stratified morphology, similar to that found in native cartilage. The second goal was to further increase chondrocyte proliferation, and extracellular matrix (ECM) accumulation. To achieve this, constructs were grown in a bioreactor with media supplemented with 14 mM sodium bicarbonate (NaHCO3). Constructs cultivated in the bioreactor with NaHCO3 supplementation exhibited a significant (p<0.05) increase in ECM accumulation (a 98-fold increase in glycosaminoglycans and a 25-fold increase in collagen content), cell proliferation (a 13-fold increase), and thickness (a 28-fold increase) compared to all other conditions (static and reactor without NaHCO3 supplementation).

The third goal was to engineer cartilage constructs with as little cells as possible, reducing donor site morbidity. From the results obtained, it was evident that the monolayer constructs outperformed all the other constructs (pellet, biopsy, and minced).

The final goal was to understand the underlying reason for the increased proliferation. First, I investigated if there were any differences present in intracellular pH (pHi) and intracellular buffering capacity. Second, I determined the role of extracellular pH (pHe) on cell proliferation. In an effort to accurately achieve this, I, for the first time, have reported on measuring pHi of chondrocytes while still in culture (2D and 3D cultures) using a confocal microscope. This study demonstrated the importance of extracellular environments, such as pHe, extracellular buffering capacity, and the presence of carbon dioxide and bicarbonate ions for chondrocyte proliferation.